Alcoholic liver disease (ALD) is a prominent source of morbidity and mortality in the United States. ALD is a progressive disease encompassing hepatic steatosis, steatohepatitis, fibrosis and cirrhosis. It is now widely accepted the molecular mechanisms underlying ALD are multifactorial including compromised antioxidant systems resulting in overproduction of reactive oxidant species having the potential to damage hepatocellular lipids, proteins and nucleic acids. The oxidant-mediated modification of lipids and the resulting modification of proteins critical for hepatocellular homeostasis are important mechanistic events contributing to initiation and/or progression of ALD. The general working hypothesis underlying the proposed experiments is that protein modification occurring as a consequence of lipid peroxidation plays a significant role in the pathogenesis of ALD. During the previous funding period, significant progress has been made in the development of state-of-the-art proteomic approaches for isolating, identifying and functionally characterizing proteins modified by the lipid peroxidative products 4-hydroxynonenal (4-HNE) and 4-oxononenal. These proteomic approaches will be used to test this hypothesis in the following three specific aims: Experiments in Aim 1 will characterize the functional consequences of 4-HNE modification on proteins targets previously identified including urea cycle enzymes, F0F1 ATPase and ER stress proteins. Studies proposed in Aim 2 will identify the 4-HNE- and 4-ONE-modified proteome in the Total Enteral Nutrition (TEN) model of alcohol-induced steatosis, steatohepatitis or fibrosis to identify protein modifications that are mechanistically involved in these progressive stages of ALD. These studies will employ administration of the hepatoprotectant N-acetylcysteine to identify changes in the 4-HNE or 4-ONE-modified proteomes responsive to this antioxidant. Experiments proposed in Aim 3 will employ this same animal model to characterize the production and cytotoxicity of autoantibodies against 4-HNE and 4-ONE-modified host proteins during ALD characterized by steatosis, steatohepatitis and fibrosis. Experiments proposed in conjunction with this aim use immunoisolation techniques and LC-MS/MS mass spectrometry to identify the specific host proteins which function as autoantigens. Collectively, the mechanistic information derived from these experiments will provide new insight into novel therapeutic strategies to attenuate and or reverse ALD. 7. Project Narrative: The mechanisms by which chronic ethanol ingestion damages the liver are multiple and interactive. The advancement in our knowledge of this disease will evolve from systematic investigations of individual mechanisms which contribute to this complex and progressive disease. It is now clear that oxidative stress is an important component of ALD. Lipid peroxidation is one consequence of oxidative stress resulting in the production of products lipid aldehydes such as 4-HNE and 4-ONE that are important factors in liver injury associated with ALD. The overall goal of this renewal application is to extend our investigation of the mechanisms by which these endogenous compounds are involved in the pathophysiology of early (steatosis) inflammatory (steatohepatitis) as well as late stages of ALD, including fibrosis. Public Health Relevance: The cytochromes P450 are a large superfamily of proteins that in humans are responsible for oxidizing a vast array of compounds, including many endogenous compounds such as steroids, bile acids, vitamins, and xenobiotics such as drugs, anesthetics, solvents, and compounds in our diet. The proposed research will lead to a better understanding of how these enzymes function, especially with their redox partners, cytochrome b5 and cytochrome P450 reductase.